This invention consists of an improved method for the use of fiber optics as a means to evaluate human and animal bodily tissues.
Fluorescence detection from tissue is increasingly being used as a diagnostic probe to either detect early cancer, to monitor treatment dose in photodynamic therapy, to monitor fluorescent drug pharmacokinetics, or to assay for fluorescent reporters of metabolic processes. It is well recognized that the pigments of a tissue may reduce the intensity of a light signal, so that fluorescence measurements from one sample cannot be directly compared to another without some type of empirical calibration. Also variation in blood flow and tissue heterogeneity can confound a series of fluorescence measurements, due to the fact that the excitation and fluorescent light are multiply scattered. At present there is no standard method for quantitative tissue fluorescence measurement in vivo, and many investigators rely upon the use of radioactive labeling or tissue extraction methods to yield quantitative drug uptake measurements. A new type of fiber optic probe has been developed and tested here in tissue-simulating phantoms, which is insensitive to the local background absorbing molecules, and detects fluorescence signals which are linearly proportional to the fluorophore concentration. This design allows quantitative measurement of fluorophore concentration for certain applications, and minimizes the need for expensive and time consuming tissue extraction or radio-labeling methods. This fiber optic method may allow non-invasive quantification of fluorescence for any applications where the inter-sample variations of tissue optical properties affect the measurements.
The prior art teaches that most tissue fluorescence measurements use large core fiber bundles, or separate source-detector fibers which allow the excitation and emission light to be multiply scattered in the tissue, and hence the fluorescence intensity is affected by the long pathlength of travel in the tissue. One practical approach to quantitative tissue fluorescence measurements has been to use relative fluorescence as a ratio at two different wavelengths, or from comparing the fluorescence intensity of two different species present in the tissue. These approaches suffer from problems in calibration and wavelength variations in the tissue optical properties; hence, the measurements must be carefully interpreted. Alternatively, several researchers have developed models of fluorescence propagation in tissue to predict fluorescence signals from multiple scattering samples such as tissue for a given fluorophore concentration. By applying time-resolved or frequency-domain instrumentation, the lifetimes and concentrations of a fluorophore can be calculated, assuming that the light propagation in the tissue can be accurately modeled by diffusion theory, or with Monte Carlo scattering models. The diffusion theory approach has the constraint that the region of tissue sampled needs to be sufficiently large and homogenous. The Monte Carlo approach is generally used to exactly model the tissue geometry with boundaries and changes in optical properties at the microscopic level. This latter approach can be very successful, but is limited to applications where the tissue properties and layers are well understood. Both of these methods require some apriori knowledge about the tissue in order to quantitatively estimate the fluorophore concentration from the detected signal.
Alternatively fluorescence measurements can be obtained from tissue using very small sample volumes, and by confining the region of tissue probed to a diameter smaller than the average scattering length, where the effects of tissue scatter and absorption can be minimized. This latter approach has the benefit of significantly minimizing the tissue effects upon the detected fluorescence signal so that intensity variations due to the tissue intrinsic chromophores are not detected. Practical implementation of this approach requires limiting the detected volume of tissue to a region smaller than the typical mean free path for scattering in tissue, which is approximately 100 microns (a micron is equal to one millionth of a meter). While limiting the volume of tissue sampled can significantly lower the detected fluorescence signal, current light detectors can be used to quantify fluorescence from sub-micromolar levels of some photosensitizers. This concept was demonstrated using confocal detection both in tissue phantoms and excised tissues. This study extends that work to examine the use of small fiber optics to measure minimally scattered fluorescence from tissue-simulating media in this manner. To realize this experimentally, a new fiber optic bundle is demonstrated which allows detection of fluorescence from confined regions of tissue, while sampling from multiple locations simultaneously to maximize the detected signal. Applications of this system will likely be in non-invasively measuring luminescent drugs taken up in vivo, and monitoring pharmacokinetics of fluorescently labeled metabolites non-invasively.
A new design for a fiber optic bundle was demonstrated to measure fluorescence signals from tissue, where the intensity of the signal is not significantly affected by the medium""s absorption and scattering coefficients, and hence only depends upon the fluorophore""s properties. Monte Carlo simulations of light scattering were used to design and verify the results obtained. The fiber optic bundle was tested on tissue-simulating phantoms and compared to the use of a standard non-imaging fiber optic bundle. The new bundle was composed of 30 individual 100 xcexcm (xe2x80x9cxcexcmxe2x80x9d is used as the abbreviation for diameter in microns) fibers, where each fiber was separated by approximately 1 mm (xe2x80x9cmmxe2x80x9d is used as the abbreviation for millimeters) from each other fiber on the end of the bundle which contacts the tissue surface and are without separation on the opposite end. This design allows integration of the signal over several locations, while maintaining localized sampling from regions smaller than the average mean free scattering path of the tissue. The bundle was tested by measuring fluorescence signals from tissue-simulating solutions containing a fluorescent compound. These studies demonstrate that the new bundle reduces the effect of the intrinsic absorption in the medium, allowing detection of fluorescence that is linearly proportional to the fluorophore concentration.